A Laccase Gene Reporting System That Enables Genetic Manipulations in a Brown Rot Wood Decomposer Fungus Gloeophyllum trabeum

ABSTRACT Brown rot fungi are primary decomposers of wood and litter in northern forests. Relative to other microbes, these fungi have evolved distinct mechanisms that rapidly depolymerize and metabolize cellulose and hemicellulose without digesting the more recalcitrant lignin. Its efficient degradative system has therefore attracted considerable attention for the development of sustainable biomass conversion technologies. However, there has been a significant lack of genetic tools in brown rot species by which to manipulate genes for both mechanistic studies and engineering applications. To advance brown rot genetic studies, we provided a gene-reporting system that can facilitate genetic manipulations in a model fungus Gloeophyllum trabeum. We first optimized a transformation procedure in G. trabeum, and then transformed the fungus into a constitutive laccase producer with a well-studied white rot laccases gene (from Trametes versicolor). With this, we built a gene reporting system based on laccase gene’s expression and its rapid assay using an 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) indicator dye. The laccase reporter system was validated robust enough to allow us to test the effects of donor DNA’s formats, protoplast viability, and gene regulatory elements on transformation efficiencies. Going forward, we anticipate the toolset provided in this work would expedite phenotyping studies and genetic engineering of brown rot species. IMPORTANCE One of the most ubiquitous types of decomposers in nature, brown rot fungi, has lacked robust genetic tools by which to manipulate genes and understand its biology. Brown rot fungi are primary decomposers in northern forests helping recycle the encased carbons in trees back to ecosystem. Relative to other microbes, these fungi employ distinctive mechanisms to disrupt and consume the lignified polysaccharides in wood. Its decay mechanism allows fast, selective carbohydrate catabolization, but without digesting lignin—a barren component that produces least energy trade back for fungal metabolisms. Thus, its efficient degradative system provides a great platform for developing sustainable biotechnologies for biomass conversions. However, progress has been hampered by the lack genetic tools facilitating mechanistic studies and engineering applications. Here, the laccase reporter system provides a genetic toolset for genetic manipulations in brown rot species, which we expect would advance relevant genetic studies for discovering and harnessing the unique fungal degradative mechanisms.

genetic transformation system for the same fungus. It undermines some of the novelty. I understand Microbiology Spectrum is not novelty-focused, but the relevant literature must be considered. On Line 16 the authors state, "However, there has been no tractable brown-rot strain in which to..." but the below paper seems to call this into question. Also, this paper seems to undermine another claim of this study whereby they "express an endogenous (G. trabeum) laccase candidate gene (Gtlcc3) under...". The reporter system claimed in the submitted manuscript is laccase-based and works partly because on line 191 the authors state, "laccase ... and its absence of enzyme activities in brown rot fungi....".
Apologies if there is a misunderstanding about species names etc, which can frequently be revised. Line 1: the title must include the name of the fungus, and also adding that it is a laccase-based reporter system would be more informative to the reader.
Line 86: It should be stated clearly in M&Ms that the strain is a monokaryon.
Line 152: It states that mitotic stability was verified by five successive transfers on hygromycin. Did this include a type of ON/OFF selection sub-culturing, i.e., subculture onto hyg, then sub-culture onto the plate without hyg, and then sub-culture to another plate with hyg. This can help verify if the transformant is pure and also that the resistance gene will not be "lost".
Line 154: Did the authors also perform a Southern blot to confirm integration into the basidiomycete genome? I can see they did for the laccase constructs later.
Line 216: The viability of the protoplasts is described. But did the authors also check the authenticity of the protoplasts e.g., if the protoplasts were diluted in water, and plated on medium without an osmotic stabilizer, there should not be any colonies if all of what were considered protoplasts were real/authentic protoplasts. This is more of a problem when germinating spores are used for protoplasting as a non-germinated spore can be mistaken for a protoplast.
Line 261: "was sent" is not the best expression.
Line 424: The authors should discuss more how the laccase would compare as a reporter gene to luciferase, GFP, etc. Especially considering that background laccase activity is partly assumed not to be a problem because the laccase-like genes in G. trabeum are not expressed. I think GFP can also be similarly quick to laccase if one uses an epi-fluorescent binocular type microscope for screening plates. I think it is worth noting that the laccases may be developmentally regulated, such as during fructification, but this will not be a problem with monokaryon. Fig. 4, part (E), here the relative protoplast regeneration % seem quite high but in line 216 it states that the protoplast regeneration rate was 1-2%. If the values are relative to one treatment, instead I think it is better to show the actual regeneration percentages.
I think depositing the vectors described in this work in the Addgene database can be very useful for other researchers, but it is of course not essential for publication.
Did the authors check how many nuclei are present in the protoplasts, such as by Hoechst stain or SYBR green? It is not directly relevant to this study but can be useful for future research on gene deletions. e.g., if the protoplasts are multinucleate, it can be difficult to purify transformants. The type of transformation work described by the authors does not confirm that their transformants are genetically pure, but it is probably unlikely that wild type cells are maintained if the antibiotic concentration is high enough.
Sorry if I have missed this, was a blender ever used in the mycelia preparation? Blenders such as Waring blenders are commonly used with wood-rotting fungi.
I'm curious, is this work also the subject of any patent applications, as transformations of brown rots also has commercial applications?
Reviewer #2 (Comments for the Author): The authors report the construction of gene manipulation system in the model brown-rot fungus Gloeophyllum trabeum. The easy-to-detect laccase was successfully used as a reported gene. The research was well designed and clearly presented in the manuscript. The system is expected to be used for studying the mechanisms of lignocellulose degradation as well as other biological processes in this important fungus. The following issues should be addressed. 1. The preparation of protoplasts is key for DNA transformation. Did the authors try the use of only one of the two lysing enzymes, or lysing enzymes of other sources? Such details/notes in transformation would be very helpful for other researchers in this field. 2. As I know the genetic manipulation tools in some basidiomycetous fungi are still quite limited, despite the report of successful expression of commonly used reporter genes. I suggest the authors provide more discussions on such difficulties or challenges in future development of gene targeting and gene editing methods in Gloeophyllum trabeum (and maybe basidiomycetous fungi). 3. How to understand the different sizes of the colonies of transformants in Fig. 2B? 4. Line 99. I would not agree with that the ABTS-medium was used for the screening of transformants. It seems this plate was only used for phenotypic analysis of transformants screened out using hygromycin B. 5. Line 110. The accession number of pMD-1 or the sequence of HygR on this plasmid should be provided. 6. Line 138. Novoenzyme should be Novozymes. 7. Line 143. Better use "production" to replace "productivity". 8. Line 185. subscript T is usually used in CT. 9. Line 268. Why mention or highlight the nitrogen content of HMM? 10. Figs. 1 and 2. gdp should be gpd. Kb should be kb (also correct in the main text). 11. Fig. 2D. Images from different experiments should not be spliced together. A space can be left between them.

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First, we appreciate the editor's and reviewers' constructive feedback and contributions to our manuscript. We believe this review process will have helped improve this manuscript and advance our next-step brown rot phenotyping research in this emerging model species. According to reviewers' comments, we have revised the manuscript and provide the following responses, point-by-point:

Responses to Reviewer #1:
Comments: The authors develop a transformation system and laccase-based reporter system for a brown rot fungus G. trabeum. The authors have a track record of developing resources for the wood-rotting fungal community and are experts with brown rots and this particular brown rot. The work seems technically sound with sufficiently detailed methods, but there is one major issue related to the previous literature that I have concerns about. The following study does not seem to be cited by the authors? This must be rectified by the authors. This study describes a genetic transformation system for the same fungus. It undermines some of the novelty. I understand Microbiology Spectrum is not novelty-focused, but the relevant literature must be considered. On Line 16 the authors state, "However, there has been no tractable brown-rot strain in which to..." but the below paper seems to call this into question. Also, this paper seems to undermine another claim of this study whereby they "express an endogenous (G. trabeum) laccase candidate gene (Gtlcc3)  Response: This is a very helpful citation addition, and to avoid any overstatements, we have toned down our language in some lines, with an example, as follows: "However, there has been a significant lack of genetic tools in brown-rot species by which to…" in revised lines 16-17, 28, and throughout the manuscript. In terms of this referred paper, we feel it is not reducing the novelties of the gene reporting system we developed, but instead it does a very good job supporting working within this species. It is a nice citation addition. In our paper, we primarily focused on the development of a laccase-based gene reporting system and its applications for optimizing transformation methods. We feel this is prominently distinct from the referred paper where the "molecular breeding" was the main goal. With this context, we reported a large number of optimized details on how to establish a more robust genetic transformation system, which we believe will further support others' research in this field. Given its clear support to our research, we have cited this suggested paper revised lines 317-. The use of laccase as the reporter gene has been validated and stated from several aspects throughout the manuscript. We have summarized these here to highlight: 1) the absence of native laccase genes' expression in wild-type G. trabeum ATCC 11539 strain by qPCR and RNA-seq ( Fig. 2E; Zhang et al., 2019, mBio), 2) the absence of WT laccase activities under variable C and N conditions (Fig. 4), and 3) we have been using a heterologous laccase gene obtained from a white-rot fungus Trametes versicolor as the reporter gene. In addition, this absence of WT laccase expression is also true in the reviewer referred citation, where the native laccase gene Gtlcc3 has